The Pet127 protein is a mitochondrial 5'-to-3' exoribonuclease from the PD-(D/E)XK superfamily involved in RNA maturation and intron degradation in yeasts.

Pet127 is a mitochondrial protein found in multiple eukaryotic lineages, but absent from several taxa, including plants and animals. Distant homology suggests that it belongs to the divergent PD-(D/E)XK superfamily which includes various nucleases and related proteins. Earlier yeast genetics experiments suggest that it plays a nonessential role in RNA degradation and 5' end processing. Our phylogenetic analysis suggests that it is a primordial eukaryotic invention that was retained in diverse groups, and independently lost several times in the evolution of other organisms. We demonstrate for the first time that the fungal Pet127 protein in vitro is a processive 5'-to-3' exoribonuclease capable of digesting various substrates in a sequence nonspecific manner. Mutations in conserved residues essential in the PD-(D/E)XK superfamily active site abolish the activity of Pet127. Deletion of the PET127 gene in the pathogenic yeast Candida albicans results in a moderate increase in the steady-state levels of several transcripts and in accumulation of unspliced precursors and intronic sequences of three introns. Mutations in the active site residues result in a phenotype identical to that of the deletant, confirming that the exoribonuclease activity is related to the physiological role of the Pet127 protein. Pet127 activity is, however, not essential for maintaining the mitochondrial respiratory activity in C. albicans.

Mitotic chromosome condensation requires phosphorylation of the centromeric protein KNL-2 in C. elegans.

Centromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1 in vitro, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation, and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans. This article has an associated First Person interview with the first author of the paper.

Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line.

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing.

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.

Loss of histone H3.3 results in DNA replication defects and altered origin dynamics in C. elegans.

Histone H3.3 is a replication-independent variant of histone H3 with important roles in development, differentiation, and fertility. Here, we show that loss of H3.3 results in replication defects in Caenorhabditis elegans embryos at elevated temperatures. To characterize these defects, we adapt methods to determine replication timing, map replication origins, and examine replication fork progression. Our analysis of the spatiotemporal regulation of DNA replication shows that despite the very rapid embryonic cell cycle, the genome is replicated from early and late firing origins and is partitioned into domains of early and late replication. We find that under temperature stress conditions, additional replication origins become activated. Moreover, loss of H3.3 results in altered replication fork progression around origins, which is particularly evident at stress-activated origins. These replication defects are accompanied by replication checkpoint activation, a delayed cell cycle, and increased lethality in checkpoint-compromised embryos. Our comprehensive analysis of DNA replication in C. elegans reveals the genomic location of replication origins and the dynamics of their firing, and uncovers a role of H3.3 in the regulation of replication origins under stress conditions.

Adaptations for centromere function in meiosis.

The aim of mitosis is to segregate duplicated chromosomes equally into daughter cells during cell division. Meiosis serves a similar purpose, but additionally separates homologous chromosomes to produce haploid gametes for sexual reproduction. Both mitosis and meiosis rely on centromeres for the segregation of chromosomes. Centromeres are the specialized regions of the chromosomes that are attached to microtubules during their segregation. In this review, we describe the adaptations and layers of regulation that are required for centromere function during meiosis, and their role in meiosis-specific processes such as homolog-pairing and recombination. Since female meiotic divisions are asymmetric, meiotic centromeres are hypothesized to evolve quickly in order to favor their own transmission to the offspring, resulting in the rapid evolution of many centromeric proteins. We discuss this observation using the example of the histone variant CENP-A, which marks the centromere and is essential for centromere function. Changes in both the size and the sequence of the CENP-A N-terminal tail have led to additional functions of the protein, which are likely related to its roles during meiosis. We highlight the importance of CENP-A in the inheritance of centromere identity, which is dependent on the stabilization, recycling, or re-establishment of CENP-A-containing chromatin during meiosis.

H3.3K27M-induced chromatin changes drive ectopic replication through misregulation of the JNK pathway in C. elegans.

Substitution of lysine 27 with methionine in histone H3.3 is a recently discovered driver mutation of pediatric high-grade gliomas. Mutant cells show decreased levels and altered distribution of H3K27 trimethylation (H3K27me3). How these chromatin changes are established genome-wide and lead to tumorigenesis remains unclear. Here we show that H3.3K27M-mediated alterations in H3K27me3 distribution result in ectopic DNA replication and cell cycle progression of germ cells in Caenorhabditis elegans. By genetically inducing changes in the H3.3 distribution, we demonstrate that both H3.3K27M and pre-existing H3K27me3 act locally and antagonistically on Polycomb Repressive Complex 2 (PRC2) in a concentration-dependent manner. The heterochromatin changes result in extensive gene misregulation, and genetic screening identified upregulation of JNK as an underlying cause of the germcell aberrations. Moreover, JNK inhibition suppresses the replicative fate in human tumor-derived H3.3K27M cells, thus establishing C. elegans as a powerful model for the identification of potential drug targets for treatment of H3.3K27M tumors.

Differential Expression of Histone H3.3 Genes and Their Role in Modulating Temperature Stress Response in Caenorhabditis elegans.

Replication-independent variant histones replace canonical histones in nucleosomes and act as important regulators of chromatin function. H3.3 is a major variant of histone H3 that is remarkably conserved across taxa and is distinguished from canonical H3 by just four key amino acids. Most genomes contain two or more genes expressing H3.3, and complete loss of the protein usually causes sterility or embryonic lethality. Here, we investigate the developmental expression patterns of the five Caenorhabditis elegans H3.3 homologs and identify two previously uncharacterized homologs to be restricted to the germ line. Despite these specific expression patterns, we find that neither loss of individual H3.3 homologs nor the knockout of all five H3.3-coding genes causes sterility or lethality. However, we demonstrate an essential role for the conserved histone chaperone HIRA in the nucleosomal loading of all H3.3 variants. This requirement can be bypassed by mutation of the H3.3-specific residues to those found in H3. While even removal of all H3.3 homologs does not result in lethality, it leads to reduced fertility and viability in response to high-temperature stress. Thus, our results show that H3.3 is nonessential in C. elegans but is critical for ensuring adequate response to stress.

Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function.

Small RNAs called PIWI-interacting RNAs (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by the PIWI protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing PIWI proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway.

Modulation of Activity of Ultrashort Lipopeptides toward Negatively Charged Model Lipid Films.

Because of the increasing resistance of pathogens to commonly used antibiotics, there is an urgent need to find alternative antimicrobial compounds with different mechanisms of action. Among them, lipopeptides are recognized as promising candidates. In this work, the Langmuir technique and atomic force microscopy were employed to investigate the interactions of two novel lipopeptides with negatively charged phospholipid membranes, which served as a simplified model of inner membrane of Gram-negative bacteria. Lipid films contained phosphatidylethanolamine and phosphatidylglycerol extracts from E. coli bacteria. Lipopeptides were composed of palmitoyl chain covalently coupled to N-terminus of peptide with Trp-Lys-Leu-Lys amino acid sequence and the conformation of third residue was either d-Leu or l-Leu. It was found that chirality of leucine strongly affects interfacial behavior of these compounds, which was ascribed to the difference in effective size of the peptide portion of the molecules. Although the lipopeptides were the same in terms of amino acid sequence, charge, and identity of lipophilic chain, the experiments revealed that the barrier for their insertion into the lipid membrane is significantly different. Namely, it was lower for lipopeptide containing d-Leu residue. We have also found that insertion of the lipopeptides into the model membranes strongly alters lateral distribution of the membrane components and leads to its substantial fluidization. The dynamics of reorganization was noticeably faster in the presence of lipopeptide with smaller size of peptide moiety, i.e., containing d-Leu. It proves that effective size of the peptide headgroup is an important factor determining lipopeptide activity toward the lipid membranes.

The transcriptome of Candida albicans mitochondria and the evolution of organellar transcription units in yeasts.

BACKGROUND: Yeasts show remarkable variation in the organization of their mitochondrial genomes, yet there is little experimental data on organellar gene expression outside few model species. Candida albicans is interesting as a human pathogen, and as a representative of a clade that is distant from the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Unlike them, it encodes seven Complex I subunits in its mtDNA. No experimental data regarding organellar expression were available prior to this study. METHODS: We used high-throughput RNA sequencing and traditional RNA biology techniques to study the mitochondrial transcriptome of C. albicans strains BWP17 and SN148. RESULTS: The 14 protein-coding genes, two ribosomal RNA genes, and 24 tRNA genes are expressed as eight primary polycistronic transcription units. We also found transcriptional activity in the noncoding regions, and antisense transcripts that could be a part of a regulatory mechanism. The promoter sequence is a variant of the nonanucleotide identified in other yeast mtDNAs, but some of the active promoters show significant departures from the consensus. The primary transcripts are processed by a tRNA punctuation mechanism into the monocistronic and bicistronic mature RNAs. The steady state levels of various mature transcripts exhibit large differences that are a result of posttranscriptional regulation. Transcriptome analysis allowed to precisely annotate the positions of introns in the RNL (2), COB (2) and COX1 (4) genes, as well as to refine the annotation of tRNAs and rRNAs. Comparative study of the mitochondrial genome organization in various Candida species indicates that they undergo shuffling in blocks usually containing 2-3 genes, and that their arrangement in primary transcripts is not conserved. tRNA genes with their associated promoters, as well as GC-rich sequence elements play an important role in these evolutionary events. CONCLUSIONS: The main evolutionary force shaping the mitochondrial genomes of yeasts is the frequent recombination, constantly breaking apart and joining genes into novel primary transcription units. The mitochondrial transcription units are constantly rearranged in evolution shaping the features of gene expression, such as the presence of secondary promoter sites that are inactive, or act as "booster" promoters, simplified transcriptional regulation and reliance on posttranscriptional mechanisms.